Method of replicating viruses in suspension cultures of dog kidney cells

ABSTRACT

Animal cells are described which can be infected by viruses and which are adapted to growth in suspension in medium free of animal-derived components, such as serum-free medium. Processes for the replication of viruses in cell culture using these cells are furthermore described, as well as vaccines which contain the viruses or antigenic portions thereof obtainable by the process.

FIELD OF THE INVENTION

The present invention relates to animal cells which can be infected byviruses and are adapted to growth in suspension in medium free ofanimal-derived components, such as serum-free medium, and to processesfor the replication of viruses in cell culture using these cells. Thepresent invention further relates to the viruses obtainable by theprocess described and to vaccines which contain viruses of this type orconstituents thereof.

BACKGROUND

Many vaccines including influenza vaccine for the treatment of humansand animals consist of one or more virus strains which have beenreplicated in embryonated hens' eggs. These viruses are isolated fromthe allantoic fluid of infected hens' eggs and their antigens are usedin vaccines as intact virus particles, as virus particles disintegratedby detergents and/or solvents, as chemically or physically inactivatedviruses, or as isolated, defined virus proteins as in subunit vaccines.The viruses are often inactivated by processes known to the personskilled in the art. The replication of live attenuated viruses, whichare tested in experimental vaccines, is also carried out in embryonatedhens' eggs.

The use of embryonated hens' eggs for vaccine production is time-,labor- and cost-intensive. The eggs, from healthy flocks of hensmonitored by veterinarians, have to be incubated before infection,customarily for 12 days. Before infection, the eggs have to be selectedwith respect to living embryos, as only these eggs are suitable forvirus replication. After infection the eggs are again incubated,customarily for 2 to 3 days. The embryos still alive at this time arekilled by subjecting them to a cold environment, and the allantoic fluidis then obtained from the individual eggs by aspiration. By means oflaborious purification processes, substances from the hen's egg thatlead to undesired side effects of the vaccine are separated from theviruses, and the viruses are concentrated. As eggs are not sterile(pathogen-free), it is additionally necessary to remove and/or toinactivate pyrogens and all pathogens that are possibly present.

Viruses of other vaccines, such as, for example, rabies viruses, mumps,measles, rubella, polio viruses, tick bourne encephalits viruses such asFriihsommer-Meningo Ecephalitis (FSME) virus can be replicated in cellcultures. As cell cultures originating from tested cell banks arepathogen-free and, in contrast to hens' eggs, are defined virusreplication systems that (theoretically) are available in almostunlimited amounts, they make possible economical virus replication undercertain circumstances even in the case of influenza viruses. Moreover,the isolation and replication of influenza viruses in eggs leads to aselection of certain phenotypes, of which the majority differ from theclinical isolate. In contrast to this is the isolation and replicationof the viruses in cell culture, in which no passage-dependent selectionoccurs (Oxford, J. S. et al., J. Gen. Virology 72 (1991), 185-189;Robertson, J. S. et al., J. Gen. Virology 74 (1993) 2047-2051). For aneffective vaccine, therefore, virus replication in cell culture ispreferred.

It is known that influenza viruses can be replicated in cell cultures.Beside hens' embryo cells and hamster cells (BHK21-F and HKCC), MDBK andMDCK cells have been described as suitable cells for the in-vitroreplication of influenza viruses (Kilbourne, E. D., in: Influenza, pages89-110, Plenum Medical Book Company-New York and London, 1987). Aprerequisite for a successful infection is the addition of proteases tothe infection medium, preferably trypsin or similar serine proteases, asthese proteases extracellularly cleave the precursor protein ofhemagglutinin [HA₀] into active hemagglutinin [HA₁ and HA₂]. Onlycleaved hemagglutinin leads to the adsorption of the influenza viruseson cells with subsequent virus assimilation into the cells (Tobita, K.et al., Med. Microbiol. Immunol., 162 (1975), 9-14; Lazarowitz, S. G. &Choppin, P. W., Virology, 68 (1975) 440-454; Klenk, H.-D. et al.,Virology 68 (1975) 426-439) and thus to a further replication cycle ofthe virus in the cell culture.

U.S. Pat. No. RE 33,164 (from U.S. Pat. No. 4,500,513), which is whollyincorporated by reference herein, described the replication of influenzaviruses in cell cultures of adherently grown CLDK cells (or, “aCLDKcells”). The constraining requirement of growing these cells adherentlyplaces a limitation on the yield of cells that can be grown and alsoconsequently places a limitation on the yield of virus that can beharvested for formulation in a vaccine.

Moreover, growing virus in adherent (or, substrate-dependent) cellsrequires steps not necessary when the cells can be grown in suspension.After cell proliferation, the nutrient medium is removed and freshnutrient medium is added to the cells with infection of the cells withinfluenza viruses taking place simultaneously or shortly thereafter. Agiven time after the infection, protease (e.g. trypsin) is added inorder to obtain an optimum virus replication. The viruses are harvested,purified and processed to give inactivated or attenuated vaccine.

Economical influenza virus replication as a prerequisite for vaccineproduction cannot be accomplished, however, using the methodologydescribed in U.S. RE 33,164, as the change of media, the subsequentinfection as well as the addition of trypsin, which is carried outlater, necessitates opening the individual cell culture vessels severaltimes and is thus very labor-intensive. Furthermore, the danger ofcontamination of the cell culture by undesirable micro-organisms andviruses increases with each manipulation of the culture vessels. Yetanother disadvantage with this system is that serum (including withoutlimitation fetal calf serum, fetal bovine serum (fbs), newborn calfserum or bovine serum) is necessary for the growth of the cells. Serumcontains trypsin inhibitors that interfere with viral yield.

A more cost-effective alternative is cell proliferation in systems wherethe cells do not need to be grown adherently to the culture container oron the surface of micro carriers. U.S. Pat. No. 6,656,720, which iswholly incorporated by reference herein, provides an example of one suchmethod wherein MDCK cells that are grown in suspension are infected withinfluenza virus. However, additional cell lines and methodologies areneeded that provide alternative means of growing viruses to increaseefficiencies and reduce overall costs.

Hence, there is a need for additional cell lines that can be cultured inmedium that is free of animal-derived components (e.g., serum-freemedium or animal protein-free medium) to reduce the risk associated withuse of animal by-products (e.g., bovine serum) and to eliminate theexpense of such animal by-products. Furthermore, there is also a generalneed to eliminate the necessity of substrate-dependent growth (e.g.,T-flask, roller bottle or micro carriers) and to have suspensioncultures instead. Suspension cultures have numerous advantages oversubstrate-dependent growth including cost savings, higher cell densitiesand greater virus yields.

SUMMARY OF THE INVENTION

The present invention is directed to a method of replicating virus in aculture of sCLDK cells comprising: a) inoculating a suspension of sCLDKcells with a virus, thereby infecting the sCLDK cells; b) allowing thevirus to reproduce in the infected sCLDK cells; and c) harvesting thevirus from the suspension culture of sCLDK cells. The virus can be aninfluenza virus, such as a human influenza virus, an avian influenzavirus, an equine influenza virus, a swine influenza virus, a canineinfluenza virus, or a feline influenza virus. Without limitation, theinfluenza virus can be an H1 influenza virus, an H2 influenza virus, anH3 influenza virus, an H5 influenza virus or an H7 influenza virus.Without limitation, the influenza virus can be an H5N1 influenza virus,an H3N8 influenza virus, an H1N1 influenza virus, an H3N2 influenzavirus, an H2N3 influenza virus, an H7N8 influenza virus or an H3N1influenza virus. In an embodiment of the invention, shortly beforeinoculating, simultaneously with inoculating, or shortly afterinoculating, a protease to cleave the precursor protein of hemagglutininis added to the suspension of sCLDK cells. The protease can be trypsin.

The present invention is also directed to a process of adaptingsubstrate-dependent CLDK cells for growth in suspension comprising a)inoculating a sample of substrate-dependent CLDK cells in a mediumcomprising serum substitutes; b) growing the cells in suspension in themedium; c) serially passaging the CLDK cells in suspension in freshbatches of the medium; and d) weaning the CLDK cells in suspension offof the serum substitutes by reducing the amount of the serum substitutesin the medium to zero. This process is also referred to as “theadaptation process.” The medium can be serum-free. The medium can alsobe free of any animal derived components.

In one embodiment of the adaptation process, the cells are seriallypassaged in step (c) until the seven day growth factor is at least fromabout 3 to 20, preferably greater than about 5, more preferably greaterthan about 7, most preferably greater than about 10. After the averagegrowth factor for 3 to 15 serial passages of step (c) has achieved suchvalues, the CLDK cells in suspension can be weaned off of the serumsubstitutes over the subsequent 3 to 6 passages. As a non-limitingexample, CLDK cells are serially passaged in suspension until theaverage growth factor of at least 5-10 passages in suspension is greaterthan about 5, at which point they are weaned off of the serum substituteover the subsequent 6 or fewer passages. Preferably, the CLDK cells areserially passaged in suspension until the average growth factor of atleast 10 passages in suspension is greater than about 8, at which pointthey are weaned off of the serum substitute over the subsequent 6 orfewer passages. Alternatively, the CLDK cells are serially passaged insuspension until the average growth factor of at least 5 passages insuspension is greater than about 12, at which point they are weaned offof the serum substitute over the subsequent 6 or fewer passages.

The CLDK cells can successfully be weaned off of the serum substitutesin the adaptation process providing that the 7 day growth factor of thepassage immediately prior to the first weaning passage is at least 5.Preferably, the 7 day growth factor of the last passage immediatelyprior to the weaning passage is at least 7, more preferably at least 10,even more preferably at least about 13.

The present invention is also directed to a method of continuouslypropagating sCLDK cells in suspension comprising: a) inoculating sCLDKcells into a medium; b) growing the sCLDK cells in suspension for aperiod of from about 4 to 10 days; c) transferring a sample of culturedmaterial from (b) into fresh cell-free medium; d) and repeating (b) and(c) for a period of continuous growth. The medium can be serum-free. Themedium can be animal protein-free. The sample can be transferred to thefresh cell-free medium without the prior addition of any protease. Thecell density after transferring the sample according to (c) can be atleast 3×10⁵ cells/mL, and is preferably at least 5×10⁵ cells/mL. ThesCLDK cells can have a growth factor greater than 3 over a 4-10 dayperiod, preferably a growth factor greater than about 5, most preferablygreater than about 10, over a 4-10 day period.

The present invention is also directed to sCLDK cells capable of growingin suspension obtainable by the adaptation process. As described above,the adaptation process is the process of adapting substrate-dependentCLDK cells for growth in suspension comprising a) inoculating a sampleof substrate-dependent CLDK cells in a medium comprising serumsubstitutes; b) growing the cells in suspension in the medium; c)serially passaging the CLDK cells in suspension in fresh batches of themedium; and d) weaning the CLDK cells in suspension off of the serumsubstitutes by reducing the amount of the serum substitutes in themedium to zero. The medium can be serum-free. The serum substitutes canbe animal-protein free. The present invention is also directed to use ofsuch sCLDK cells obtained by this process for replicating virus. Thepresent invention is also directed to use of such virus replicated insuch sCLDK cells obtained by this process in the manufacture of avaccine.

The present invention is also directed to a composition comprising cellculture medium and sCLDK cells.

The present invention also relates to a method of making a vaccinecomprising a) inoculating medium with sCLDK cells; b) growing the sCLDKcells in suspension in the medium, thereby generating a suspension ofsCLDK cells; c) incubating the suspension of sCLDK cells with virusthereby generating a suspension of infected sCLDK cells; d) allowing thevirus to reproduce in the infected sCLDK cells; e) harvesting orisolating the virus; and f) mixing the harvested or isolated virus withone or more pharmaceutically acceptable carrier thereby making avaccine.

DETAILED DESCRIPTION

The present invention relates to making available cells and processesthat make possible simple and economical virus replication in cellculture. The invention thus relates to animal cells that can be infectedby viruses and that are adapted to growth in suspension in serum-free oranimal protein-free medium. It was found that it is possible with theaid of cells of this type to replicate viruses in cell culture in asimple and economical manner. Because the cells are adapted for growthin suspension, production batches yield greater numbers of cells andsubsequently higher titers of virus. A greater yield of virus reducesoverall costs of production. A further advantage is that the consumptionof media is markedly decreased, thereby reducing total media costs. Alsoimportantly, an advantage of the present invention is growth of cells(and subsequent replication of viruses) in medium that is free of anyanimal derived components, such as bovine serum or fetal calf serum,thereby eliminating the risk of various pathogens including withoutlimitation TSE (transmissible spongiform encephalopathy).

Moreover, by the use of the cells according to the invention, on the onehand a change of medium before infection to remove serum can bedispensed with and on the other hand the addition of protease (whereneeded) can be carried out simultaneously with the infection. On thewhole, only a single opening of the culture vessel for infection withviruses is necessary, whereby the danger of the contamination of thecell cultures is drastically reduced. The expenditure of effort thatwould be associated with the change of medium, the infection and thesubsequent protease addition is furthermore decreased.

The cells used according to the invention are derived fromsubstrate-dependent Cutter Laboratory Dog Kidney (CLDK) cells. As usedherein, “substrate-dependent CLDK cells” and like terms are usedinterchangeably with “adherent CLDK cells,” “aCLDK cells” or like terms.aCLDK cells are different from suspension CLDK cells. Whereas the formergrow adherently, the latter grow in suspension, such as in a shakerflask or other large-scale production container including withoutlimitation disposable or non-disposable bioreactors or biocontainers.aCLDK cells require a surface on which to grow, such as the surface of aroller bottle or microcarriers or beads added to the medium. aCLDK cellsdo not grow in suspension. When inoculated into medium in a shaker flaskplaced on an oscillating platform, aCLDK cells may maintain someviability, but do not grow. Rather, aCLDK cells tend to clump together.

Suspension CLDK cells are also termed “sCLDK” cells or “sCLDK-SF” cellsto indicate that they grow in a serum-free media. sCLDK cells arederived from aCLDK cells by passaging through serum-free medium insuspension as described herein. On account of these properties and theirability to serve as host cells for replicating viruses, sCLDK cells aresuitable for economical replication of viruses in cell culture by meansof a simple and cost-effective process. In contrast to aCLDK cells,sCLDK cells do grow in a shaker flask placed on an oscillating platformor in other suspension growth means. Not only do sCLDK cells maintainviability in such suspended growth conditions, but they can multiply innumber without aggregating or clumping as is the case with aCLDK cells.

As described further below, sCLDK cells have several advantages overaCLDK cells. First, unlike aCLDK cells, sCLDK cells grow in suspension.Unlike aCLDK cells, sCLDK cells have a growth factor of at least three(3) when passed into fresh media for growth in suspension over a 4-10day period. In an embodiment of the invention, the sCLDK cells describedherein grow in suspension by a factor of greater than 3, preferablygreater than 5, in a 4-10 day period. More preferably, the sCLDK cellsdescribed herein grow in suspension by a factor of greater than about 6,most preferably greater than about 10, in a 4-10 day period.

Unlike aCLDK cells, sCLDK cells are also able to grow continuously insuspension. sCLDK cells can grow continuously in suspension by a factorof greater than 3, preferably greater than 5, when passed into freshmedia every 4-10 days. More preferably, the sCLDK cells described hereincan grow continuously in suspension by a factor of greater than about 6,most preferably greater than about 10, when passed into fresh mediaevery 4-10 day period.

Another advantage of sCLDK cells over aCLDK cells is that the former cangrow in the absence of serum or other animal derived media supplements.Unlike aCLDK cells, sCLDK cells on average at least triple when passedinto fresh media containing no serum or other animal derived mediasupplements over a 4-10 day period. In an embodiment of the invention,the sCLDK cells described herein grow in media containing no serum orother animal derived media supplements by a factor of greater than 3,preferably greater than 5, in a 4-10 day period. More preferably, thesCLDK cells described herein grow when passed into fresh mediacontaining no serum or other animal derived media supplements by afactor of greater than about 6, most preferably greater than about 10,in a 4-10 day period.

Unlike aCLDK cells, sCLDK cells are also able to grow continuously inmedia containing no serum or other animal derived components. sCLDKcells can grow continuously in media containing no serum or other animalderived components by a factor of greater than 3, preferably greaterthan 5 when passed into fresh media every 4-10 days. More preferably,the sCLDK cells described herein can grow continuously in mediacontaining no serum or other animal derived components by a factor ofgreater than about 6, most preferably greater than about 10, when passedinto fresh media every 4-10 days.

Hence, an advantage of sCLDK cells is their ability to grow continuouslyin suspension in media containing no serum or other animal derivedcomponents. sCLDK cells can grow continuously in suspension in mediacontaining no serum or other animal derived components by a factor ofgreater than 3, preferably greater than 5, when passed into fresh mediaevery 4-10 days. More preferably, the sCLDK cells described herein cangrow continuously in suspension in media containing no serum or otheranimal derived components by a factor of greater than about 6, mostpreferably greater than about 10, when passed into fresh media every4-10 day period.

As used herein, “growth factor” or like terms refer to themultiplicative value by which an initial cell population has grown overa period of time. Hence, a growth factor of 2 or 3 indicates that thecell density has respectively doubled or tripled relative to thestarting cell density when cells were passed (i.e., inoculated orplanted) into the fresh media. A growth factor of 10 or 20 indicatesthat the cell density has respectively multiplied by a factor of 10 or20 relative to the starting cell density when the cells were passed intothe fresh media. A growth factor of 1 or less indicates that the celldensity did not increase or that the cell population has declined.

The present invention therefore also relates to a process for thereplication of viruses in cell culture in which cells according to theinvention are used. In particular the process that comprises thefollowing steps: i) proliferation of the cells according to theinvention described above in serum-free medium in suspension; ii)infection of the cells with viruses; iii) culturing of the infectedcells; and iv) isolation of the replicated viruses. Where titer can beincreased as in the case of influenza virus, protease can be addedshortly before, simultaneously to or shortly after infection. In onenon-limiting embodiment of the invention, sCLDK cells are grown in aserum free media to replicate influenza virus. The skilled artisan isaware of other viruses that can be replicated in the sCLDK cellsdescribed herein, and is not limited to replication of influenzaviruses.

The present invention is also directed to a method of replicating virusin a culture of sCLDK cells comprising: a) inoculating a suspension ofsCLDK cells with a virus, thereby infecting the sCLDK cells; b) allowingthe virus to reproduce in the infected sCLDK cells; and c) harvestingthe virus from the suspension culture of sCLDK cells. The virus can beharvested through centrifugation, filtration or other mechanical orbiochemical separation means of the infected cell suspension. Hence,harvested virus can include virus in the presence of sCLDK cells orsCLDK cell debris. Harvested virus can also include virus in the absenceof sCLDK cells or sCLDK cell debris.

The cells according to the invention can preferably be cultured in mediafree of animal derived components (e.g., serum-free media, animalprotein-free media) known to the person skilled in the art. Non-limitingexamples of such media include Iscove's medium, ultra CHO medium(BioWhittaker), EX-CELL™ MDCK serum-free medium (JRH Biosciences,Lenexa, Kans.)). Other serum-free media that may be used according tothe present invention include EX-CELL™ 520 medium (JRH Biosciences,Lenexa, Kans.) and HyQ PF CHO medium (Hyclone, Logan, Utah). Otheranimal protein-free media that may be used according to the presentinvention include EX-CELL™ 302 medium (JRH Biosciences, Lenexa, Kans.),HyQ PF CHO MPS medium (Hyclone, Logan, Utah) and Rencyte BHK medium(Medicult, Jyllinge, Denmark). Other soy- or yeast-based animalprotein-free media that may be used according to the invention includethose described in U.S. Pat. No. 7,160,699 or U.S. Published PatentApplications No. 2003/0203448, 2004/0077086, 2004/0087020, 2004/0171152,2005/0089968, 2006/0094104, 2006/0286668, which are all herebyincorporated by reference in their entirety. Suitable culture vesselsthat can be employed in the course of the process according to theinvention are all vessels known to the person skilled in the art.

Serum substitutes can be added to the animal-derived component-freemedia. Such substitutes are themselves free of any animal-derivedcomponents, yet may contain recombinant animal proteins expressed incells that were preferably cultured in media free of animal-derivedcomponents. Serum substitutes can contain recombinant growth factors,transferrin substitutes or recombinant transferrin substitutes,synthetic hormones and/or other recombinant proteins. Serum substitutesare free of any adventitious viruses because the recombinant proteincomponents are manufactured in a controlled environment in pathogen-freeor viral-free cells. Hence, serum substitutes have no virus or TSE(transmissible spongiform encephalopathy) risk. Serum substitutes arepreferably used to adapt adherent CLDK cells for growth in suspension(i.e., transform aCLDK cells to sCLDK cells). However, such substitutescan also be used for culturing sCLDK cells in suspension for growth ofvirus according to the present invention. A non-limiting example of aserum substitute includes LIPUMIN™ serum substitute (PAA LaboratoriesGmbH, Pasching, Austria).

The temperature for the proliferation of the cells before infection asin the case with influenza viruses is preferably 37° C. Culturing forproliferation of the cells can be carried out in a perfusion system,e.g. in a stirred vessel fermenter, using cell retention systems knownto the person skilled in the art, such as, for example, centrifugation,filtration, spin filters and the like, using batch processes or usingother techniques well known to the skilled artisan.

The cells are in this case preferably proliferated for 2 to 18 days,particularly preferably for 3 to 11 days. Exchange of the medium iscarried out in the course of this, increasing from 0 to approximately 1to 3 volumes per day. The cells are proliferated up to very high celldensities in this manner, preferably up to approximately 2×10⁷ cells/ml.Perfusion rates during culture in a perfusion system can be regulatedboth via the cell count, the content of glucose, glutamine or lactate inthe medium and via other parameters known to the person skilled in theart. In the case of infection with influenza viruses, about 85% to 99%,preferably 93 to 97%, of the fermenter volume is transferred with cellsto a further fermenter. The cells remaining in the first fermenter canin turn be mixed with medium and replicated further in the perfusionsystem. In this manner, continuous cell culture for virus replication isavailable.

In a preferred embodiment of the process according to the invention, thepH of the culture medium used can be regulated during culturing and isin the range from pH 6.6 to pH 7.8, preferably in the range from pH 6.8to pH 7.3.

Furthermore, the pO₂ value is advantageously regulated in this step ofthe process and is preferably between 25% and 95%, in particular between35% and 60% (based on the air saturation). According to the invention,the infection of the cells cultured in suspension is preferably carriedout when the cells in the batch process have achieved a cell density ofabout 8 to 25×10⁵ cells/ml or about 5 to 20×10⁶ cells/ml in theperfusion system.

In a further preferred embodiment, the infection of the cells in thecase of influenza viruses is carried out at an m.o.i. (multiplicity ofinfection) of about 0.0001 to 10, preferably of 0.002 to 0.5. Theaddition of the protease, which brings about the cleavage of theprecursor protein of hemagglutinin [HA₀] and thus the adsorption of theviruses on the cells, can be carried out according to the inventionshortly before, simultaneously to or shortly after the infection of thecells with influenza viruses. If the addition is carried outsimultaneously with the infection, the protease can either be addeddirectly to the cell culture to be infected or, for example, as aconcentrate together with the virus inoculate. The protease ispreferably a serine protease, and particularly preferably trypsin.

In a preferred embodiment, trypsin is added to the cell culture to beinfected to a final concentration of 1 to 200 μg/ml, preferably 5 to 50μg/ml, and particularly preferably 5 to 30 μg/ml in the culture medium.During the further culturing of the infected cells according to theinvention, trypsin reactivation can be carried out by fresh addition oftrypsin in the case of the batch process or in the case of the perfusionsystem by continuous addition of a trypsin solution or by intermittentaddition. In the latter case, the trypsin concentration is preferably inthe range from 1 μg/ml to 80

After infection, the infected cell culture is cultured further toreplicate the viruses, in particular until a maximum cytopathic effector a maximum amount of virus antigen can be detected. Preferably, theculturing of the cells is carried out for 2 to 10 days, in particularfor 3 to 7 days. The culturing can in turn preferably be carried out inthe perfusion system or in the batch process.

In a further preferred embodiment, the cells can be cultured at atemperature of 30° C. to 37° C. in an incubator set at 5-15% CO₂, mostpreferably around 10% CO₂.

The culturing of the cells after infection as in the case of influenzaviruses is in turn preferably carried out at regulated pH and pO₂. ThepH in this case is preferably in the range of from 6.6 to 7.8,particularly preferably from 6.8 to 7.2, and the pO₂ in the range offrom 25% to 150%, preferably from 30% to 75%, and particularlypreferably in the range of from 35% to 60% (based on the airsaturation).

During the culturing of the cells or virus replication of the process, asubstitution of the cell culture medium with freshly prepared medium,medium concentrate or with defined constituents such as amino acids,vitamins, lipid fractions, phosphates etc. for optimizing the antigenyield is also possible.

After infection as in the case with influenza viruses, the cells caneither be slowly diluted by further addition of medium or mediumconcentrate over several days or can be incubated during furtherperfusion with medium or medium concentrate decreasing fromapproximately 1 to 3 to 0 fermenter volumes/day. The perfusion rates canin this case in turn be regulated by means of the cell count, thecontent of glucose, glutamine, lactate or lactate dehydrogenase in themedium or other parameters known to the person skilled in the art.

A combination of the perfusion system with a fed-batch process isfurther possible. In a preferred embodiment of the process, theharvesting and isolation of the replicated virus (e.g., influenza virus)is carried out within 2 to 10 days, preferably 3 to 7 days, afterinfection. To do this harvesting, for example, the cells or cellresidues are separated from the culture medium by means of methods knownto the person skilled in the art, for example by centrifugation,separators or filters. Following such steps, the concentration of thevirus present in the culture medium is carried out by methods known tothe person skilled in the art, such as, for example, gradientcentrifugation, filtration, precipitation and the like.

The invention further relates to influenza viruses that are obtainableby a process according to the invention. They can be harvested andformulated by known methods to give a vaccine for administration tohumans or animals. The immunogenicity or efficacy of a vaccinecomprising the influenza viruses obtained can be determined by methodsknown to the person skilled in the art, e.g., by means of the protectionimparted in the loading experiment or as antibody titers of neutralizingantibodies. The determination of the amount of virus or antigen producedcan be carried out, for example, by the determination of the amount ofhemagglutinin according to methods known to the person skilled in theart. It is known, for example, that cleaved hemagglutinin binds toerythrocytes of various species, e.g. to chicken erythrocytes. Thismakes possible a simple and rapid quantification of the viruses producedor of the antigen formed.

Thus the invention also relates to vaccines that contain virusobtainable from the process according to the invention. Vaccines of thistype can optionally contain the additives customary for vaccines, inparticular substances that increase the immune response, i.e.,adjuvants, e.g. hydroxide of various metals, carbomers, constituents ofbacterial cell walls, oils or saponins, and customary pharmaceuticallytolerable excipients.

The viruses can be present in the vaccines as intact virus particles, inparticular as live attenuated viruses. For this purpose, virusconcentrates are adjusted to the desired titer and either lyophilized orstabilized in liquid form.

In a further embodiment, the vaccines according to the invention cancontain disintegrated, inactivated or intact but inactivated viruses.For this purpose, the infectiousness of the virus is destroyed by meansof chemical and/or physical methods (e.g., by detergents orformaldehyde). The vaccine is then adjusted to the desired amount ofantigen and after possible admixture with adjuvants or after possiblevaccine formulation, dispensed, for example, in liposomes, microspheresor other “slow release” formulations.

In a further preferred embodiment, the vaccines according to theinvention can finally be present as subunit vaccine, i.e. they cancontain defined, isolated virus constituents, preferably isolatedproteins of the influenza or other virus. These constituents can beisolated from the virus by methods known to the person skilled in theart.

Furthermore, influenza or other viruses obtained by the processaccording to the invention can be used for diagnostic purposes. Thus thepresent invention also relates to diagnostic compositions that containinfluenza or other viruses according to the invention or constituents ofsuch viruses, if appropriate, in combination with additives customary inthis field and suitable detection agents.

The following examples are merely illustrative, and not limiting to theremainder of this disclosure in any way.

EXAMPLES Example 1A Adaptation of CLDK Cells for Serum-Free or SerumSubstitute-Free Suspended Growth

Cutter Laboratory Dog Kidney (CLDK) cells are anchorage dependent, andhence require a substrate or surface on which to grow. Suitablesubstrates include the interior surfaces of containers such as T-flasksor roller bottles, or upon the surface of beads or microcarriers thatcan be added to a culturing container. Typically, these cells formmonolayers on the substrate, and can be grown in static culture androller bottle. These cells require serum to grow and are epithelial inmorphology.

Two ampoules of Cutter Laboratories aCLDK cells were thawed, and thentransferred to two 4 mL cryotubes such that there was 1 mL of thawedmaterial in each tube. 1 mL of supplemented EX-CELL™ MDCK serum-freemedium was added to each cryotube. “Supplemented EX-CELL™ MDCKserum-free medium” as used herein refers to a mixture of EX-CELL™ MDCKserum-free medium (SAFC Biosciences, Lenexa, Kans.) supplemented withL-glutamine (20 mLs of 200 mM solution/L of medium) and gentamicin (0.5mL of 100 g/mL solution for every liter of medium). After allowing thetwo cryotubes to sit undisturbed for 3-5 minutes, all of the materialwas transferred to two 15 mL centrifuge tubes. Approximately 8 mLs ofsupplemented EX-CELL™ MDCK serum-free medium was added to each 15 mlcentrifuge tube. These two tubes were then allowed to sit undisturbedfor 3-5 minutes, after which time the two tubes were centrifuged for tenminutes at about 1,000 rpms (500×g). The media supernatant was discardedand the pelleted cells were resuspended in a total of 20 mLs ofsupplemented EX-CELL™ MDCK serum-free medium. The resuspended cells weretransferred to a 125 mL shaker flask fitted with a 0.2μ vented cap(Corning Inc., Corning, NY), to which 30 mLs of additional supplementedEX-CELL™ MDCK serum-free medium was added to bring the total volume to50 mLs. These cells correspond to passage no. 1 in Table 1 below andwere placed on an orbital shaker plate in a 37° C. water jacketedincubator with a 10% CO₂ feed for a week.

Cells were passed on a weekly basis two more times (passages 2 and 3 inTable 1) without serum or serum substitutes and in shaker flasks so asto require any growth to be in suspension. However, as shown in Table 1,the highest recorded growth factor for the aCLDK cells under theseconditions was less than 3 per 7-day period. The subsequent 13 passagesalso included 1% Pluronic® F68 surfactant (Invitrogen Corp., Carlsbad,Calif.) in the culturing media. During this time, cells remained highlyviable but continued to have a growth factor that was (on average) lessthan 2 per 7-day period, as shown in Table 1. The next 14 passages alsoincluded 1% Lipumin™ ADCF (animal derived component free) serumsubstitute (PAA Laboratories GmbH, Pasching, Austria) in the media.During these passages, the average growth factor (as shown in Table 1)was greater than 12 and the cells were >95% viable. In the followingpassage, cells were weaned to 0.5% Pluronic® F68 surfactant and 0.5%Lipumin™ ADCF serum substitute. In the next passage, cells were weanedto 0.25% Pluronic® F68 surfactant and 0.25% Lipumin™ ADCF serumsubstitute. The subsequent 6 passages had the cells completely free ofPluronic® F68 surfactant and Lipumin™ ADCF serum substitute. Duringthese last 6 passages, the cells had a growth factor on average greaterthan 17 (as shown in Table 1) and continued to have high viability.

Table 1 shows the passage history described in this example.Supplemented EX-CELL™ MDCK serum-free medium was used in every passagewith the indicated amount of Pluronic® F68 surfactant or Lipumin™ ADCFserum substitute as shown, for a total volume of 50 mLs. Each passagewas into a 125 mL shaker flask and placed on an orbital shaker plate ina 37° C. water jacketed incubator with a 10% CO₂ feed for a week. The“Cell Plant Density” refers to the concentration of cells in the 50 mLsof medium at the beginning of the passage (i.e., the beginning of theweek) and is given in cells/mL. After a week, the cells were countedfrom a sample of material to determine the “Cell count/mL.” The “7-DayGrowth Factor” was determined by dividing the “Cell count/mL” value bythe “Cell Plant Density” value.

Table 1: Passage History to Adapt aCLDK Cells to sCLDK-SF Cells

% Pluronic ® % Lipumin ™ Cell Plant Cell 7-Day F68 ADCF Serum Densitycount/ Growth Passage Surfactant Substitute (cells/mL) mL Factor 1 0 0NA* NA NA 2 0 0 NA 2.10 × 10⁵ NA 3 0 0 2.00 × 10⁵ 4.89 × 10⁵ 2.45 4 1 02.00 × 10⁵ 2.24 × 10⁵ 1.12 5 1 0 2.00 × 10⁵ 2.15 × 10⁵ 1.08 6 1 0 5.00 ×10⁵ 6.39 × 10⁵ 1.28 7 1 0 5.00 × 10⁵ 6.54 × 10⁵ 1.31 8 1 0 5.00 × 10⁵5.13 × 10⁵ 1.03 9 1 0 5.00 × 10⁵ 6.92 × 10⁵ 1.38 10 1 0 5.00 × 10⁵ 6.96× 10⁵ 1.39 11 1 0 5.00 × 10⁵ 7.19 × 10⁵ 1.44 12 1 0 5.00 × 10⁵ 5.57 ×10⁵ 1.11 13 1 0 5.00 × 10⁵ 7.24 × 10⁵ 1.45 14 1 0 5.00 × 10⁵ 5.51 × 10⁵1.10 15 1 0 5.00 × 10⁵ 4.74 × 10⁵ 0.95 16 1 0 4.00 × 10⁵ 6.92 × 10⁵ 1.7317 1 1 5.00 × 10⁵ 3.61 × 10⁵ 0.72 18 1 1 3.00 × 10⁵ 1.62 × 10⁶ 5.40 19 11 5.00 × 10⁵ 2.10 × 10⁶ 4.20 20 1 1 5.00 × 10⁵ 5.63 × 10⁶ 11.26 21 1 15.00 × 10⁵ 8.79 × 10⁶ 17.58 22 1 1 5.00 × 10⁵ 5.21 × 10⁶ 10.42 23 1 15.00 × 10⁵ 3.25 × 10⁶ 6.50 24 1 1 5.00 × 10⁵ 3.63 × 10⁶ 7.26 25 1 1 5.00× 10⁵ 7.21 × 10⁶ 14.42 26 1 1 5.00 × 10⁵ 1.17 × 10⁷ 23.40 27 1 1 5.00 ×10⁵ 8.68 × 10⁶ 17.36 28 1 1 5.00 × 10⁵ 1.02 × 10⁷ 20.37 29 1 1 5.00 ×10⁵ 8.50 × 10⁶ 17.00 30 1 1 5.00 × 10⁵ 8.08 × 10⁶ 16.16 31 0.5 0.5 5.00× 10⁵ 6.80 × 10⁶ 13.60 32 0.25 0.25 5.00 × 10⁵ 3.19 × 10⁷ 63.80 33 0 05.00 × 10⁵ 5.30 × 10⁵ 1.06 34 0 0 5.00 × 10⁵ 9.96 × 10⁶ 19.92 35 0 05.00 × 10⁵ 9.10 × 10⁶ 18.20 36 0 0 5.00 × 10⁵ 1.03 × 10⁷ 20.50 37 0 05.00 × 10⁵ 3.42 × 10⁶ 6.84 38 0 0 5.00 × 10⁵ 1.99 × 10⁷ 39.80 *NA: notavailable

From the cells of the 38th passage, a small cell stock (15-20 cryovials)was frozen in ampoules and stored in liquid nitrogen (LN) with 10% DMSO(Sigma-Aldrich Co., St. Louis, Mo.). The designation was changed tosCLDK-SF, referring to “s” as suspension and “-SF” as serum free duringthis process. No serum was utilized in the freezing of this cell line.

Approximately five months after freezing vials after the 38th passage,two of the frozen vials were thawed and placed in a shaker flask. Thesecells were grown for one week in a 125 mL shaker flask (passage no. 39).These cells were then scaled up (over two more passages) to 4400 mLusing eleven 1L Nalgene shaker flasks, each fitted with a 0.2μ ventedcap. Day four (96 hours) into passage no. 41, log-phase (activelygrowing) cells were harvested, 10% DMSO was added and glass ampouleswere filled and sealed with an automated ampoule filling and sealingmachine under HEPA filtered air. The master cell stock bank wasinspected for proper seals, labeled and frozen in liquid nitrogen.Several frozen samples were thawed and screened for potentialcontaminates (mold and bacterial contamination). Screening revealed nobacterial or mold growth after 21 days in tryptone soy broth andtryptose phosphate broth at 28° C. and 37° C., respectively.

Example 1B Adaptation of aCLDK Cells for Serum-Free or SerumSubstitute-Free Suspended Growth

The above CLDK adaptation procedure was subsequently repeated andcompleted with fewer passages. The following table summarizes theculture media and observed growth for this subsequent procedure.

TABLE 2 Passage History to Adapt aCLDK Cells to sCLDK-SF Cells %Pluronic ® % Lipumin ™ Cell Plant Cell 7-Day F68 ADCF Serum Densitycount/ Growth Passage Surfactant Substitute (cells/mL) mL Factor 1 0 0NA 7.94 × 10⁵ NA 2 0 0 5.00 × 10⁵ 5.95 × 10⁵ 1.19 3 1 1 5.00 × 10⁵ 2.65× 10⁵ 0.53 4 1 1 2.50 × 10⁵ 1.08 × 10⁶ 4.32 5 1 1 5.00 × 10⁵ 4.15 × 10⁵0.83 6 1 1 3.00 × 10⁵ 4.45 × 10⁵ 1.48 7 1 1 4.00 × 10⁵ 2.35 × 10⁵ 0.59 81 1 2.00 × 10⁵ 8.34 × 10⁵ 4.17 9 1 1 5.00 × 10⁵ 2.09 × 10⁶ 4.18 10 1 15.00 × 10⁵ 1.67 × 10⁶ 3.34 11 1 1 5.00 × 10⁵ 3.35 × 10⁶ 6.70 12 1 1 5.00× 10⁵ 3.25 × 10⁶ 6.50 13 1 1 5.00 × 10⁵ 3.90 × 10⁶ 7.80 14 1 1 5.00 ×10⁵ 7.10 × 10⁶ 14.20 15 1 1 5.00 × 10⁵ 7.28 × 10⁶ 14.56 16 1 1 5.00 ×10⁵ 6.84 × 10⁶ 13.68 17 1 1 5.00 × 10⁵ 6.68 × 10⁶ 13.36 18 0.5 0.5 5.00× 10⁵ 8.10 × 10⁶ 16.20 19 0.25 0.25 5.00 × 10⁵ 7.37 × 10⁶ 14.74 20 0.1250.125 5.00 × 10⁵ 2.76 × 10⁶ 5.52 21 0 0 5.00 × 10⁵ 6.87 × 10⁶ 13.74 22 00 5.00 × 10⁵ 7.42 × 10⁶ 14.84 23 0 0 5.00 × 10⁵ 7.80 × 10⁶ 15.60 24 0 05.00 × 10⁵ 1.00 × 10⁷ 20.00

Example 1C Procedure for Recovering sCLDK-SF MCS Cells from LiquidNitrogen Storage and Optimum Growth Propagation

1L of supplemented EX-CELL™ MDCK serum-free medium was prepared using anaseptic technique. Thereafter, 2-3 mLs of cells from the frozen ampoulesof passage 41 of Example 1A were retrieved from liquid nitrogen andallowed to thaw. The thawed ampoules were sprayed with 70% alcohol andallowed to dry. A sterile ampoule snapper was used to break open allvials. Thereafter, a sterile pipette was used to transfer an ampoule ofcells to a small sterile tube with cap (sterile 4.5 mL cryovial orequivalent). This step was repeated for the second and third ampoules.

With a sterile pipette, 1.0 mL of supplemented EX-CELL™ MDCK serum freemedium was added to each tube. After these tubes containing thecell-medium mixture sat for approximately 3-5 minutes, a sterile pipettewas used to transfer cell-medium mixture from one small sterile tube toa 15 mL centrifuge tube (Falcon or equivalent). This step was repeatedfor the second and third small tubes containing cell-media mixture.Approximately 8.0 mLs of supplemented EX-CELL™ MDCK Serum free mediumwas added to each tube. After allowing all 15 mL centrifuge tubescontaining 10 mLs each of cell-medium mixture to sit for 5-10 minutes,the tubes were centrifuged for 10 minutes at approximately 500×g. Themedia was then poured out of the centrifuge tubes while keeping thepellet(s). Approximately 5 mLs of supplemented EX-CELL™ MDCK serum freemedium was added to each centrifuge tube with a sterile pipette. Thepellets were then re-suspended in the centrifuge tubes. The re-suspendedcell-media mixtures from all tubes were transferred into one 125 mLshaker flask fitted with a 0.2 μl vented cap. Approximately 40 mLs ofsupplemented EX-CELL™ MDCK serum free media was then added to the 125 mLshaker flask, which was then placed on an orbital shaker (100-110 RPM)inside a 3TC incubator set at 10% CO₂. The cells were incubated four toseven days. Seven days yielded the highest cell growth factors without asubstantial loss in viability. Cell and viability count were performedusing an automated mammalian cell counter.

Example 1D Procedure for Passing sCLDK-SF MCS Cells for GrowthPropagation

The following procedure was optimized for continuous growth of sCLDK-SFcells. This process begins after recovering cells as in Example 1C, andcan be repeated every 7 days to maintain continuous cell growth. Cellsare grown in supplemented EX-CELL™ MDCK serum-free medium.

After retrieving a shaker flask containing live sCLDK-SF cells insupplemented EX-CELL™ MDCK serum free medium from the incubator, asterile pipette is used to transfer a cell sample from the shaker flaskto a sample vial. Cell and viability count is performed using anautomated or manual means of counting. Once counts are performed, thetotal volume of cells needed to plant a new shaker flask with 50 mLs ata cell density of 5.0×10⁵ cells/mL (or, 2.5×10⁷ cells/125 mL shakerflask) is calculated.

The calculated volume of cells is transferred via sterile pipette to anew 125 mL shaker flask. After adding the appropriate volume ofsupplemented EX-CELL™ MDCK serum-free medium for a total volume of 50mL, the shaker flask is placed on an orbital shaker (100-110 RPM). Cellsare maintained undisturbed at 37° C. in the incubator with 10% CO₂ for aseven day period. This process can be repeated once every 4-10 days tomaintain cells constantly. The following data is representative ofserial passages for continuous cell growth. Passages 1-16 occurredweekly, and passages 17-34 occurred every four days.

TABLE 3 Continuous Cell Growth of sCLDK-SF Over 34 Passages Cell plantdensity Cell count Growth Pass # (cells/mL) (cells/mL) Factor ViabilityMCS NA NA NA NA 1 NA 4.16 × 10⁶ NA 93.99% 2 5.00 × 10⁵ 7.72 × 10⁶ 15.4496.65% 3 5.00 × 10⁵ 3.49 × 10⁷ 69.80 99.99% 4 5.00 × 10⁵ 2.69 × 10⁶ 5.3899.66% 5 5.00 × 10⁵ 1.35 × 10⁷ 27.00 98.13% 6 5.00 × 10⁵ 1.30 × 10⁷26.00 NA 7 5.00 × 10⁵ 1.04 × 10⁷ 20.80 95.24% 8 5.00 × 10⁵ 3.86 × 10⁶7.72 98.83% 9 5.00 × 10⁵ 4.82 × 10⁶ 9.64 96.76% 10 5.00 × 10⁵ 1.36 × 10⁷27.20 98.96% 11 5.00 × 10⁵ 7.29 × 10⁶ 14.58 99.60% 12 5.00 × 10⁵ 1.22 ×10⁷ 24.40 99.52% 13 5.00 × 10⁵ 9.56 × 10⁶ 19.12 99.52% 14 5.00 × 10⁵1.17 × 10⁷ 23.40 99.64% 15 5.00 × 10⁵ 1.08 × 10⁷ 21.60 99.58% 16 5.00 ×10⁵ 1.05 × 10⁷ 21.00 99.50% 17 5.00 × 10⁵ 3.56 × 10⁶ 7.12 99.75% 18 5.00× 10⁵ 1.03 × 10⁷ 20.60 99.75% 19 5.00 × 10⁵ 1.19 × 10⁷ 23.80 99.26% 205.00 × 10⁵ 6.20 × 10⁶ 12.40 99.87% 21 5.00 × 10⁵ 1.10 × 10⁷ 22.00 99.39%22 5.00 × 10⁵ 2.99 × 10⁶ 5.98 99.66% 23 5.00 × 10⁵ 1.26 × 10⁷ 25.299.14% 24 5.00 × 10⁵ 1.53 × 10⁷ 30.60 99.96% 25 5.00 × 10⁵ 7.60 × 10⁶15.20 98.00% 26 5.00 × 10⁵ 1.34 × 10⁷ 26.80 99.32% 27 5.00 × 10⁵ 1.12 ×10⁷ 22.40 99.00% 28 5.00 × 10⁵ 8.47 × 10⁶ 16.94 99.77% 29 5.00 × 10⁵6.92 × 10⁶ 13.84 99.85% 30 5.00 × 10⁵ 9.58 × 10⁶ 19.16 99.78% 31 5.00 ×10⁵ 7.33 × 10⁶ 14.66 99.78% 32 5.00 × 10⁵ 9.26 × 10⁶ 18.52 99.83% 335.00 × 10⁵ 6.76 × 10⁶ 13.52 99.63% 34 5.00 × 10⁵ 1.13 × 10⁷ 22.60 99.81%

Example 2 Comparative Growth of aCLDK Cells to sCLDK-SF Cells inSuspension in Different Serum-Free Media

aCLDK cells were taken out of storage in liquid nitrogen and were grownin a 75 cm² flask for approximately 48 hours using 10% FBS in Eagle'sMinimum Essential Medium (EMEM). The cells were then transferred to an850 cm² roller bottle with 5% Fetal Bovine Serum and EMEM media. Thisroller was then trypsinized; cells were harvested and planted into twodifferent 125 ml shaker flasks at a cell density of 5.0×10⁵ cells/mL. Ofthese two flasks, one flask contained supplemented EX-CELL™ MDCKserum-free medium and the other flask contained EMEM medium. A thirdflask containing sCLDK-SF cells (from an available continuous stock)were planted at a cell density of 5.0×10⁵ cells/mL in supplementedEX-CELL™ MDCK serum-free medium. The three suspension flasks were thenplaced on an orbital shaker in a 37° C. water jacketed incubator andsampled daily (see results below) for cell and viability counts.

TABLE 4 Growth of sCLDK in Supplemented EX-CELL ™ MDCK Serum- FreeMedium Amount of Cell Day Plant Viability Growth Factor  0 5.00 × 10⁵not measured 1.0  1 1.28 × 10⁵ 82.81% 0.3  2 5.31 × 10⁵ 98.30% 1.0  31.16 × 10⁶ 99.48% 2.3  4 2.15 × 10⁶ 99.72% 4.3  5 4.46 × 10⁶ 99.75% 8.9 6 6.88 × 10⁶ 99.73% 13.8  7* 1.09 × 10⁷ 99.77% 21.8  8 5.35 × 10⁵99.06% 1.1  9 1.47 × 10⁶ 99.65% 2.9 10 3.30 × 10⁶ 99.99% 6.6 11 7.37 ×10⁶ 99.91% 14.7 12 1.21 × 10⁷ 99.80% 24.2 13 1.28 × 10⁷ 99.73% 25.6  14*1.55 × 10⁷ 99.80% 31.1 15 4.45 × 10⁵ 98.87% 0.9 16 8.35 × 10⁵ 99.40% 1.717 2.37 × 10⁶ 99.78% 4.7 18 6.81 × 10⁶ 99.93% 13.6 19 9.33 × 10⁶ 99.69%18.7 20 1.29 × 10⁷ 99.65% 25.9 21 1.33 × 10⁷ 99.77% 26.6 *Cells werepassed into fresh media every seven days.

TABLE 5 Growth of aCLDK in EMEM Media (No Serum) Day Amount of CellPlant Viability Growth Factor 0 5.00 × 10⁵ 1.0 1 5.30 × 10⁵ 98.11% 1.1 24.75 × 10⁵ 98.94% 1.0 3 5.05 × 10⁵ 99.00% 1.0 4 4.45 × 10⁵ 98.87% 0.9 53.55 × 10⁵ 98.59% 0.7 6 3.85 × 10⁵ 98.70% 0.8  7* 3.85 × 10⁵ 98.70% 0.88 1.75 × 10⁵ 97.14% 0.4 9 1.45 × 10⁵ 96.55% 0.3 10  1.75 × 10⁵ 97.14%0.4 11  1.90 × 10⁵ 97.36% 0.4 12  1.51 × 10⁵ 96.68% 0.3 13  1.30 × 10⁵96.15% 0.3 14* 1.06 × 10⁵ 95.28% 0.0 15  ** ** ** *Cells were passedinto fresh media every seven days. ** For this study to plant a shakerflask at proper density, a cell density must be at least 5 × 10E5, thiscell count was not high enough to plant an adequate (25-50 mL) amount ina shaker flask. Therefore these cells were discarded.

TABLE 6 Growth of aCLDK in Supplemented EX-CELL ™ MDCK Serum-Free MediumAmount of Cell Day Plant Viability Growth Factor  0 5.00 × 10⁵ 1.0  18.65 × 10⁵ 99.42% 1.7  2 9.25 × 10⁵ 99.45% 1.9  3 1.20 × 10⁶ 99.58% 2.4 4 1.28 × 10⁶ 99.45% 2.6  5 1.47 × 10⁶ 99.65% 2.9  6 1.65 × 10⁶ 99.69%3.3  7* 1.23 × 10⁶ 99.59% 2.5  8 4.45 × 10⁵ 98.87% 0.9  9 5.35 × 10⁵99.06% 1.1 10 8.95 × 10⁵ 99.44% 1.8 11 8.05 × 10⁵ 99.37% 1.6 12 7.45 ×10⁵ 99.32% 1.5 13 8.95 × 10⁵ 99.44% 1.8  14* 9.25 × 10⁵ 99.45% 1.9 155.65 × 10⁵ 99.11% 1.1 16 6.55 × 10⁵ 99.23% 1.3 17 8.65 × 10⁵ 99.42% 1.718 7.45 × 10⁵ 99.32% 1.5 19 8.05 × 10⁵ 99.37% 1.6 20 7.75 × 10⁵ 99.35%1.6 21 6.55 × 10⁵ 99.23% 1.3 *Cells were passed into fresh media everyseven days.

From this study it can be concluded that the aCLDK cells in EMEM mediumdo not grow in suspension culture. The data also shows that aCLDK insupplemented EX-CELL™ MDCK serum-free medium do not grow at a sufficientrate to be considered a viable alternative for production. Typically agrowth factor of at least 5 is required to be considered useful forproduction. Instead, these cells simply maintained their cell densityand viability. Lastly this study shows that the sCLDK-SF cells insupplemented EX-CELL™ MDCK serum-free medium grow very well serum freein suspension with an average split ratio above 10 and maintain a high(>99%) viability.

Example 3 Attempted Adaptation of Substrate-Dependent CLDK Cells forSuspended Growth in Different Media Containing Serum

Two cryovials of aCLDK cells were removed from liquid nitrogen tank andthawed. The material in one vial was resuspended in 10 mL ofsupplemented EX-CELL™ MDCK serum-free medium, further supplemented with5% fetal bovine serum (FBS). The material in the other vial wasresuspended in 10 mL of supplemented Hank's minimal essential medium(MEMH). Supplemented MEMH medium is described in U.S. Pat. No. RE 33,164(from U.S. Pat. No. 4,500,513), which is hereby wholly incorporated byreference. This medium as used in this example includes the following:

-   -   Fetal Bovine Serum (FBS), 5%    -   Non-Essential Amino Acids, 10 ml/L media    -   L-Glutamine, 10 mL/L media    -   Neomycin Sulfate, (0.3 mL of 100 mg/mL stock solution)/L media    -   Polymyxin B, 30,000 units/L media    -   Nystatin, 25,000 units/L media    -   50% Dextrose, 2.6 ml/L media    -   MEM Vitamins, 30 mL/L media

Both vials were centrifuged and the supernatant was discarded. Cellpellets were then separately resuspended in 10 mLs of their respectivemedium and then transferred to separate 25 mL tissue culture flasks with25 mL total volume of their respective medium (both media contained 5%FBS). Both flasks were placed into a 35° C. incubator.

The following day, both tissue flasks were observed under microscope andfound to be 90-100% confluent. Both flasks were trypsinized and cellswere transferred to appropriate 490 cm² roller bottles and placed in atotal volume of 150 mL of media/roller bottle. Both rollers were placedonto a roller cart (0.3 rpm) in a 35° C. incubator. Three days later,cells in both roller bottles were observed microscopically and found tobe 30% confluent. Another three days later, cell and viability countswere performed. The cells in the MEMH were found to contain 1.02×10⁷cells/mL that were 99.5% viable. The cells in the supplemented EX-CELL™MDCK serum-free medium with 5% FBS were found to contain 1.09×10⁷cells/mL that were 99.83% viable. Both roller bottles were trypsinizedand cells were passed into new 490 cm² roller bottles at a cell densityof 3.16×10⁵ cells/mL and placed in a total volume of 150 mL ofmedia/roller bottle. Both rollers were placed into a 35° C. incubator.

Four days after being in the incubator, cell and viability counts wereperformed. The material in the Hanks MEM was found to contain 1.12×10⁷cells/mL, with 99.5% of the cells being viable. The material in thesupplemented EX-CELL™ MDCK serum-free medium with 5% FBS was found tocontain 1.27×10⁷ cells/mL with 89.69% of the cells being viable. Bothroller bottles were trypsinized and the cells were passed into new 490cm² roller bottles at a cell density of 3.16×10⁵ cells/mL and placed ina total volume of 150 mL of media/roller bottle. Both rollers wereplaced into a 35° C. incubator.

Five days after being in the incubator, cell and viability counts wereagain performed. The material in the Hank's MEM was found to contain1.03×10⁷ cells/mL with 99.9% of the cells being viable. The material inthe supplemented EX-CELL™ MDCK serum-free medium with 5% FBS was foundto contain 1.72×10⁷ cells/mL with 91.91% of the cells being viable. Bothroller bottles were trypsinized and cells were passed into new 490 cm²roller bottles at a cell density of 3.16×10⁵ cells/mL and placed in atotal volume of 150 mL of media/roller bottle. Both rollers were placedback into a 35° C. incubator.

Four days after being in the incubator, cell and viability counts wereagain performed. The material in the Hanks MEM was found to contain5.95×10⁶ cells/mL with 99.2% of the cells being viable. The material inthe supplemented EX-CELL™ MDCK serum-free medium with 5% FBS was foundto contain 9.41×10⁶ cells/mL with 85.84% of the cells being viable. Bothroller bottles were trypsinized and the cells were passed into new 125mL shaker flasks at a cell density of 3.16×10⁵ cells/mL and placed in atotal volume of 50 mL of media/shaker flask. Both shaker flasks wereplaced onto an orbital shaker at 100-110 rpm in a 35° C. incubator.

Four days after being in the incubator, cell and viability counts wereagain performed. The material in the Hanks MEM was found to contain2.15×10⁵ cells/mL with 94.0% of the cells being viable. The material inthe supplemented EX-CELL™ MDCK serum-free medium with 5% FBS was foundto contain 1.38×10⁶ cells/mL with 99.6% of the cells being viable. Bothshaker flasks were placed back onto the orbital shaker at 100-110 rpm ina 35° C. incubator.

Three days after being in the incubator, cell and viability counts wereagain performed. The material in the Hanks MEM was found to contain2.00×10⁵ cells/mL (corresponding to a growth factor of 0.63) with 95.0%of the cells being viable. The material in the supplemented EX-CELL™MDCK serum-free medium with 5% FBS was found to contain 3.03×10⁶cells/mL (corresponding to a growth factor of 9.58) with 99.8% viable ofthe cells being viable. The CLDK cells in the supplemented EX-CELL™ MDCKserum-free medium with 5% FBS medium were then planted in a new 125 mLshaker flask at a cell density of 3.16×10⁵ cells/mL and placed on anorbital shaker in 35° C. incubator to determine whether the cells wouldcontinue to grow and placed in a total volume of 50 mL of media/shakerflask.

Seven days after being in the incubator, cell and viability counts wereagain performed. The CLDK cells in the supplemented EX-CELL™ MDCKserum-free medium with 5% FBS media were found to contain less than1.5×10⁵ cells/mL. The viability count could not be performed with thenucleocounter because values were too low. Visually, cells were veryclumpy, and the cell culture was discarded.

The above passage protocol is summarized in the table below. Passages 2through 5 were done in roller bottles, which is customary for culturingsubstrate-dependent cells. Below the double line, passages 6 and 7 weredone in shaker flasks to attempt adaptation of the cells for suspendedgrowth. As is evident from the table, however, the cell density droppedrapidly despite lengthier incubation periods after only 1 passage inshaker flasks. Indeed, the cells did not grow sufficiently to continuethe experiment after only one transfer into shaker flasks for thematerial in the Hank's MEM medium, and after only two transfers intoshaker flasks for the material in the supplemented EX-CELL™ MDCKserum-free medium. This data indicates that these substrate-dependentCLDK cells do not grow in suspension.

TABLE 7 Summary of Attempted Adaptation of Substrate-Dependent CLDKCells for Suspended Growth in Different Media Containing SerumIncubation Concentration period of of material culture being plantedmaterial Hank's EX-CELL Passage into fresh being MEM MDCK Number media.measured cells/mL viability cells/mL viability  2* everything 6 1.02 ×99.5 1.09 × 10⁷ cells/mL 99.83 transferred 10⁷ cells/mL from initialbreakout from cryovials 3 3.16 × 10⁵ cells/mL 4 1.12 × 99.5% 1.27 × 10⁷cells/mL 89.69% 10⁷ cells/mL 4 3.16 × 10⁵ cells/mL 5 1.03 × 99.9 1.72 ×10⁷ cells/mL 91.91 10⁷ cells/mL 5 3.16 × 10⁵ cells/mL 4 5.95 × 99.2 9.41× 10⁶ cells/mL 85.84 10⁶ cells/mL 6 3.16 × 10⁵ cells/mL 4 2.15 × 94.01.38 × 10⁶ cells/mL 99.6 10⁵ cells/mL No passage; same shaker 3 2.00 ×95.0 3.03 × 10⁶ cells/mL 99.8 flasks returned to 10⁵ cells/mLincubation. 7 3.16 × 10⁵ cells/mL 7 Not enough <1.5 × 10⁵ cells/mL notmaterial to continue measurable; with culturing. cells clumpy *The firstpassage involved steps immediately after breaking the cells out ofliquid nitrogen.

Example 4 Infection of sCLDK Cells with Influenza Virus

The sCLDK-SF cell line was infected with Canine flu (CIV,A/canine/Miami/05), a cold-adapted temperature sensitive H3N8 influenza(A/equine/2/Kentucky/1/91, described in U.S. Pat. No. 6,177,082, whichis hereby wholly incorporated by reference), and a third H3N8 influenzavirus (KY02, equine/Kentucky/02). When screened for HA, all threeharvested viruses tested returned positive HA results, with CIV and KY02showing a significant amount of titer. Titer data was not collected forthe cold-adapted temperature sensitive H3N8 influenza virus tested.

sCLDK-SF cells were grown in supplemented EX-CELL™ MDCK serum-freemedium prior to planting, most typically for 5-7 days. Cells wereharvested from shaker flasks and planted at desired densities (1.0×10⁶cells/mL), in supplemented EX-CELL™ MDCK serum-free medium media at 50.0mL per shaker flask (one flask per each virus) and incubated at 37° C.on a shaker orbital with 10% CO₂. One hour post plant, shakers wereinfected with appropriate virus and multiplicity of infection (MOI), seetable below. Trypsin is added, 1 mL per liter (50 microliters per 50mL). CIV and KY02 are incubated at 37° C., Flu-Avert at 34° C. Infectedshaker flask(s) were observed daily and percent of infection wasmonitored and recorded. Daily samples were taken and frozen at −70° C.to screen later for HA titer. Once it was determined that 90-100% ofcells were infected, cells were harvested and frozen at −70° C. Frozensamples were taken daily and tested for HA.

TABLE 8 Results: HA Testing Actual Titer Flask Virus MOI Est. titerTemp. HA* (log base 2) 1 CIV 0.01 6.6 log/ml 37° C. 1:512 6.3, 7.2 2KY02 0.01 6.0 log/ml 37° C. 1:128 6.3, 6.1, 6.5 3 Flu-Avert 0.01 4.83log/ml  34° C. 1:1024-2048 not available Control K9 Flu-Ref NA NA NA 1:64-128 not available CIN0692001 (HA control) *HA refers to thehemagglutination assay. The values reflect the greatest 2-fold dilutionof the material at which hemagglutination of red blood cells can beobserved. ** Actual titer values are reported in log base-2.

The above detailed description is intended only to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication so that others skilled in the art may adapt and apply theinvention in its numerous forms, as they may be best suited to therequirements of a particular use. This invention, therefore, is notlimited to the above embodiments, and may be variously modified.

All publications discussed herein are wholly incorporated by referenceherein.

1. A method of replicating virus in a culture of sCLDK cells comprising:a) inoculating a suspension of sCLDK cells with a virus, therebyinfecting the sCLDK cells; b) allowing said virus to reproduce in saidinfected sCLDK cells; and c) harvesting said virus from said suspensionculture of sCLDK cells.
 2. The method of claim 1, wherein said virus isan influenza virus.
 3. The method of claim 2, wherein said influenzavirus is selected from the group consisting of a human influenza virus,an avian influenza virus, an equine influenza virus, a swine influenzavirus, a canine influenza virus, and a feline influenza virus.
 4. Themethod of claim 3, wherein said influenza virus is an H3 influenzavirus, an H5 influenza virus or an H7 influenza virus.
 5. The method ofclaim 3, wherein said influenza virus is selected from the groupconsisting of an H5N1 influenza virus, an H3N8 influenza virus, and anH3N1 influenza virus.
 6. The method of claim 1, wherein shortly beforeinoculating, simultaneously with inoculating, or shortly afterinoculating, a protease to cleave the precursor protein of hemagglutininis added to the suspension of sCLDK cells.
 7. The method of claim 6,wherein said protease is trypsin.
 8. The method of claim 1, wherein saidculture of sCLDK cells was grown in serum-free medium.
 9. The method ofclaim 1, wherein said culture of sCLDK cells was grown in medium free ofany animal component derived material.
 10. A process of adaptingsubstrate-dependent CLDK cells for growth in suspension comprising a)inoculating a sample of substrate-dependent CLDK cells in a mediumcomprising one or more serum substitutes; b) growing said cells insuspension in said medium; c) serially passaging said CLDK cells insuspension in fresh batches of said medium; and d) weaning said CLDKcells in suspension off of said one or more serum substitutes byreducing the amount of said serum substitutes in said medium to zero.11. The method of claim 10, wherein said medium is serum-free.
 12. Themethod of claim 10, wherein said medium is free of animal derivedcomponents.
 13. A method of continuously propagating sCLDK cells insuspension comprising: a) inoculating sCLDK cells into a medium; b)growing said sCLDK cells in suspension for a period of from about 4 to10 days; c) transferring a sample of cultured material from (b) intofresh cell-free medium; d) and repeating (b) and (c) for a period ofcontinuous growth.
 14. The method of claim 13, wherein said medium isserum-free.
 15. The method of claim 13, wherein said medium is free ofanimal derived components.
 16. The method of claim 13, wherein saidsample is transferred to said fresh cell-free medium without the prioraddition of any protease.
 17. The method of claim 13, wherein the celldensity after transferring the sample according to (c) is at least 3×10⁵cells/mL.
 18. The method of claim 13, wherein said sCLDK cells have agrowth factor greater than about 3 over 4-10 days.
 19. sCLDK cellscapable of growing in suspension obtainable by the process of claim 10.20. (canceled)
 21. (canceled)
 22. A composition comprising cell culturemedium and sCLDK cells.